PROJECT SUMMARY/ABSTRACT The ultimate objective of this SBIR project is to develop an immunotherapy, based on our patented virus-like- vesicle (VLV) platform, for the treatment of patients infected with chronic hepatitis B virus. In Phase I, using a murine model of chronic HBV infection, we have established a Proof-of-Concept that treatment with a VLV vector expressing multiple HBV antigens (Polymerase, HBcAg and MHBs) - referred to as VLV-3xT2A - leads to a significant loss of hepatitis B surface antigen (HBsAg), a surrogate marker for HBV clearance [see Progress Report]. In fulfilling the Phase I milestones, we demonstrated (i) induction of multi-specific HBV T cells, (ii) protection from acute challenge infection, and (iii) significant reduction in HBsAg in a mouse model of persistent HBV replication.In Phase II, we will use the same murine chronic model to test an optimized prime- boost regimen to maximize the immune responses needed for clearance of the virus. In parallel, we will identify an optimal route of administration (intra-muscular or subcutaneous) that is practical for clinical use. In addition, we will perform pharmacokinetics, toxicity, and in vivo biodistribution studies in line with the feedback from the FDA?s response to our Pre-IND meeting request regarding development of VLV-3xT2A immunotherapy. The goal of this project is to develop an immunotherapy in order to achieve a functional cure, characterized by sustained loss of HBsAg (with or without HBsAg antibody seroconversion) using new VLV-based HBV therapies. The specific aims for Phase II are as follows: Aim 1: Define, optimize and validate prime and boost treatment regimens. This aim will include four tasks: Develop a prime-boost strategy and measure immune responses generated with respect to evaluating: 1) T cell response magnitude, specificity, and functional phenotype (T cell cytokine responses, cytolytic activity, etc.), and 2) antibody responses (i.e., antibody titers and antibody specificity). We will also determine the order and timing of VLV-3xT2A prime-boost administrations that results in the optimum immune response. Determine T cell responses to HBV antigens from VLV-3xT2A in the context of human MHC class I and MHC class II molecules. Compare intramuscular vs. subcutaneous routes of administration by assaying CD4+ and CD8+ T cell responses in nave mice. Evaluate the efficacy of VLV-3xT2A prime-boost regimen using the AAV-mediated model of chronic HBV. We will determine whether the immune response from the strongest prime-boost in nave mice can clear persistent HBV replication initiated in the mouse liver by AAV delivery of HBV. Aim 2: Biodistribution, pharmacokinetics, safety, toxicology, and scale-up studies. This aim involves two tasks: Conduct biodistribution and pharmacokinetic studies of VLV-3xT2A using the optimal route of administration determined in Aim 1 and conduct VLV safety and toxicology studies in mice. These results will form the basis for designing and conducting future toxicology studies by a GLP contract lab, per FDA requirements for IND submission to conduct clinical studies on VLV-3xT2A in humans. Identify conditions for scaling up VLV production and process steps amenable to current good manufacturing practice (cGMP) standards and characterize the quality and stability profiles of the optimal prime-boost VLV therapeutics.